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Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Growth of Gefitinib-resistant Non-small Cell Lung Cancer Cells by Inducing Apoptosis.

Jeong CH - J Cancer Prev (2015)

Bottom Line: The anticancer effect of USP8 inhibitor was determined by testing anchorage-dependent or independent growth of gefitinib-sensitive or resistant NSCLCs.Notably, treatment with the USP8 inhibitor was more effective in suppressing cell growth and inducing apoptosis in gefitinib-resistant HCC827GR cells than that of gefitinib-sensitive HCC827 cells.Inhibition of USP8 could be an effective strategy for overcoming gefitinib resistance in NSCLCs.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Keimyung University, Daegu, Korea.

ABSTRACT

Background: Therapeutic approach by treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib or erlotinib to non-small cell lung cancer (NSCLC) patients has been limited due to emergence of acquired drug resistance. Our study was aimed to investigate whether the inhibition of ubiquitin-specific peptidase 8 (USP8) could be an alternative strategy capable of overcoming acquired resistance to EGFR-TKIs for treatment of NSCLCs.

Methods: The anticancer effect of USP8 inhibitor was determined by testing anchorage-dependent or independent growth of gefitinib-sensitive or resistant NSCLCs. The immunoprecipitation and western blotting were conducted to check molecular interaction and signaling pathway followed by USP8 inhibition.

Results: Inhibition of USP8 induced overall degradation of oncogenic receptor tyrosine kinases including EGFR and Met, leading to a suppression of anchorage-dependent or independent cell growth of gefitinib-sensitive or resistant NSCLCs. Also, treatment with the USP8 inhibitor markedly induced apoptosis in HCC827GR cells. Notably, treatment with the USP8 inhibitor was more effective in suppressing cell growth and inducing apoptosis in gefitinib-resistant HCC827GR cells than that of gefitinib-sensitive HCC827 cells.

Conclusions: Inhibition of USP8 could be an effective strategy for overcoming gefitinib resistance in NSCLCs.

No MeSH data available.


Related in: MedlinePlus

Ubiquitin-specific peptidase (USP8) inhibitor overcomes geftinib-resistance in HCC827GR cells by inducing apoptosis. (A) Flow cytometric analysis with Annexin V staining was performed in USP8 inhibitor or gefitinib-treated HCC827 and HCC827GR cells. Flow cytometric analysis was performed using a BD FACSVersa flow cytometer and BD FACSuite software. (B) Whole cell lysates were assayed by western blot analysis after exposure to the indicated drugs (1 μM) in HCC827 and HCC827GR cells. Ge, gefitinib; Su, SU11274; USP8i, USP8 inhibitor. (C) Flow cytometric analysis with Annexin V staining was performed in drug treated HCC827 and HCC827GR cells. Error bars represent the mean ± SD. Statistical significance was determined by the Student’s t-test (*P < 0.01). Ge, gefitinib; Su, SU11274; USP8i, USP8 inhibitor. DMSO, dimethyl sulfoxide; PARP, poly adenosine diphosphate ribose polymerase.
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f3-jcp-20-57: Ubiquitin-specific peptidase (USP8) inhibitor overcomes geftinib-resistance in HCC827GR cells by inducing apoptosis. (A) Flow cytometric analysis with Annexin V staining was performed in USP8 inhibitor or gefitinib-treated HCC827 and HCC827GR cells. Flow cytometric analysis was performed using a BD FACSVersa flow cytometer and BD FACSuite software. (B) Whole cell lysates were assayed by western blot analysis after exposure to the indicated drugs (1 μM) in HCC827 and HCC827GR cells. Ge, gefitinib; Su, SU11274; USP8i, USP8 inhibitor. (C) Flow cytometric analysis with Annexin V staining was performed in drug treated HCC827 and HCC827GR cells. Error bars represent the mean ± SD. Statistical significance was determined by the Student’s t-test (*P < 0.01). Ge, gefitinib; Su, SU11274; USP8i, USP8 inhibitor. DMSO, dimethyl sulfoxide; PARP, poly adenosine diphosphate ribose polymerase.

Mentions: To determine whether anti-proliferative activity of USP8 inhibitor is resulted from the induction of apoptosis, flow-cytometry analysis with annexin V was performed. A flow cytometric analysis with Annexin V showed that treatment with the USP8 inhibitor induced early apoptosis both in gefitinib-sensitive HCC827 cells and gefitinib-resistant HCC827GR cells (Fig. 3A). Interestingly, dose-dependent treatment with 1 to 2.5 μM USP8 inhibitor in HCC827GR cells markedly induced early apoptosis at a level of 29.7% and 40.8%, respectively, but not in cells treated with 1 μM gefitinib. In HCC827 cells, however, gefitinib treatment induced early apoptosis at a level of 33%, whereas a marginal induction level was observed in USP8 inhibitor-treated cells (Fig. 3A). We next compared the total apoptosis level induced by several cancer therapeutic drugs including gefitinib, SU11274, and USP8 inhibitor in these two cell lines. Our fluorescence activated cell sorter (FACS) data revealed that the induction level of total apoptosis was evidently observed in USP8 inhibitor-treated HCC827GR cells (Fig. 3B). Its apoptotic effect was accompanied by the induction of activated caspase-3 and cleaved PARP level (Fig. 3C). Therefore, these findings clearly suggest that treatment with the USP8 inhibitor is sufficient for inducing apoptosis in gefitinib-resistant HCC827GR cells.


Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Growth of Gefitinib-resistant Non-small Cell Lung Cancer Cells by Inducing Apoptosis.

Jeong CH - J Cancer Prev (2015)

Ubiquitin-specific peptidase (USP8) inhibitor overcomes geftinib-resistance in HCC827GR cells by inducing apoptosis. (A) Flow cytometric analysis with Annexin V staining was performed in USP8 inhibitor or gefitinib-treated HCC827 and HCC827GR cells. Flow cytometric analysis was performed using a BD FACSVersa flow cytometer and BD FACSuite software. (B) Whole cell lysates were assayed by western blot analysis after exposure to the indicated drugs (1 μM) in HCC827 and HCC827GR cells. Ge, gefitinib; Su, SU11274; USP8i, USP8 inhibitor. (C) Flow cytometric analysis with Annexin V staining was performed in drug treated HCC827 and HCC827GR cells. Error bars represent the mean ± SD. Statistical significance was determined by the Student’s t-test (*P < 0.01). Ge, gefitinib; Su, SU11274; USP8i, USP8 inhibitor. DMSO, dimethyl sulfoxide; PARP, poly adenosine diphosphate ribose polymerase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384715&req=5

f3-jcp-20-57: Ubiquitin-specific peptidase (USP8) inhibitor overcomes geftinib-resistance in HCC827GR cells by inducing apoptosis. (A) Flow cytometric analysis with Annexin V staining was performed in USP8 inhibitor or gefitinib-treated HCC827 and HCC827GR cells. Flow cytometric analysis was performed using a BD FACSVersa flow cytometer and BD FACSuite software. (B) Whole cell lysates were assayed by western blot analysis after exposure to the indicated drugs (1 μM) in HCC827 and HCC827GR cells. Ge, gefitinib; Su, SU11274; USP8i, USP8 inhibitor. (C) Flow cytometric analysis with Annexin V staining was performed in drug treated HCC827 and HCC827GR cells. Error bars represent the mean ± SD. Statistical significance was determined by the Student’s t-test (*P < 0.01). Ge, gefitinib; Su, SU11274; USP8i, USP8 inhibitor. DMSO, dimethyl sulfoxide; PARP, poly adenosine diphosphate ribose polymerase.
Mentions: To determine whether anti-proliferative activity of USP8 inhibitor is resulted from the induction of apoptosis, flow-cytometry analysis with annexin V was performed. A flow cytometric analysis with Annexin V showed that treatment with the USP8 inhibitor induced early apoptosis both in gefitinib-sensitive HCC827 cells and gefitinib-resistant HCC827GR cells (Fig. 3A). Interestingly, dose-dependent treatment with 1 to 2.5 μM USP8 inhibitor in HCC827GR cells markedly induced early apoptosis at a level of 29.7% and 40.8%, respectively, but not in cells treated with 1 μM gefitinib. In HCC827 cells, however, gefitinib treatment induced early apoptosis at a level of 33%, whereas a marginal induction level was observed in USP8 inhibitor-treated cells (Fig. 3A). We next compared the total apoptosis level induced by several cancer therapeutic drugs including gefitinib, SU11274, and USP8 inhibitor in these two cell lines. Our fluorescence activated cell sorter (FACS) data revealed that the induction level of total apoptosis was evidently observed in USP8 inhibitor-treated HCC827GR cells (Fig. 3B). Its apoptotic effect was accompanied by the induction of activated caspase-3 and cleaved PARP level (Fig. 3C). Therefore, these findings clearly suggest that treatment with the USP8 inhibitor is sufficient for inducing apoptosis in gefitinib-resistant HCC827GR cells.

Bottom Line: The anticancer effect of USP8 inhibitor was determined by testing anchorage-dependent or independent growth of gefitinib-sensitive or resistant NSCLCs.Notably, treatment with the USP8 inhibitor was more effective in suppressing cell growth and inducing apoptosis in gefitinib-resistant HCC827GR cells than that of gefitinib-sensitive HCC827 cells.Inhibition of USP8 could be an effective strategy for overcoming gefitinib resistance in NSCLCs.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Keimyung University, Daegu, Korea.

ABSTRACT

Background: Therapeutic approach by treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib or erlotinib to non-small cell lung cancer (NSCLC) patients has been limited due to emergence of acquired drug resistance. Our study was aimed to investigate whether the inhibition of ubiquitin-specific peptidase 8 (USP8) could be an alternative strategy capable of overcoming acquired resistance to EGFR-TKIs for treatment of NSCLCs.

Methods: The anticancer effect of USP8 inhibitor was determined by testing anchorage-dependent or independent growth of gefitinib-sensitive or resistant NSCLCs. The immunoprecipitation and western blotting were conducted to check molecular interaction and signaling pathway followed by USP8 inhibition.

Results: Inhibition of USP8 induced overall degradation of oncogenic receptor tyrosine kinases including EGFR and Met, leading to a suppression of anchorage-dependent or independent cell growth of gefitinib-sensitive or resistant NSCLCs. Also, treatment with the USP8 inhibitor markedly induced apoptosis in HCC827GR cells. Notably, treatment with the USP8 inhibitor was more effective in suppressing cell growth and inducing apoptosis in gefitinib-resistant HCC827GR cells than that of gefitinib-sensitive HCC827 cells.

Conclusions: Inhibition of USP8 could be an effective strategy for overcoming gefitinib resistance in NSCLCs.

No MeSH data available.


Related in: MedlinePlus